A method for assaying DNA-dependent RNA polymerase II in crude extracts of Drosophila melanogaster adults: its use in identifying mutants with an altered RNA polymerase II.
A method for assaying Drosophila melanogaster adult DNa-dependent RNA polymerase II in crude extracts from as a few as two females or three males is described. Preparation of the extracts involves incubating homogenates at 25 C for 60 min and subsequent treatment with Macaloid. Eighty-five percent of the activity in the extracts is inhibited by 1 microgram/ml alpha-amanitin and this fraction is attributed to RNA polymerase II. RNA polymerase II activity in the extracts shows a good dose dependence and a partial dependence on added DNA, Mn2+, and all four ribonucleoside triphosphates. The kinetics of heat inactivation of RNA polymerase II in crude extracts could be reproducibly measured. Flies of different genotypes had different initial rates of RNA polymerase II heat inactivation. The isolation of Drosophila melanogaster alpha-amanitin-resistant mutants is also reported. Using the assay described in this paper, it appears that the basis for the resistance is an altered RNA polymerase II. The mutation has been mapped to the third chromosome by chromosome replacement.
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000032:analysis*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004351:Drug Resistance; D005260:Female; D008297:Male; D008832:Microchemistry; D009154:Mutation; D012319:RNA Polymerase II / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D013696:Temperature
D000546:Amanitins; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases